Alcohol use disorder (AUD) is a chronic relapsing disease that constitutes a major health problem for Veterans. Stress is known to be an important contributing factor to alcohol abuse and alcoholism, and this is especially relevant to Veterans given their high prevalence of co-occurring AUD and stress-related illnesses such as post- traumatic stress disorder (PTSD). Despite the significance of this problem, the complex interaction between stress and alcohol (ethanol) drinking is not fully understood. The use of animal models is critical for advancing our understanding of underlying mechanisms and providing platforms for evaluating potential new and novel treatment interventions for Veterans battling PTSD-AUD comorbidity. Recent work involving our established mouse model of ethanol dependence that involves repeated cycles of chronic intermittent ethanol (CIE) exposure led to the discovery that reduced BDNF activity in the dorsomedial prefrontal cortex (dmPFC) is a significant neuroadaptation associated with excessive drinking. During the current funding period, we extended this work to show that stress facilitates and enhances this dependence-related escalation of ethanol consumption. That is, forced swim stress (FSS) selectively enhances escalated drinking in dependent (CIE- exposed) mice while not altering more moderate ethanol intake in nondependent mice. Additionally, stress in combination with chronic ethanol exposure was shown to magnify deficits in BDNF expression in the prefrontal cortex. Further, we demonstrated that direct infusion of BDNF or viral-mediated overexpression of BDNF in the dmPFC blocked dependence (CIE)-related escalated drinking. Collectively, these data support the general tenet that reduced BDNF activity in the dmPFC plays a significant role in the ability of stress (FSS) to enhance escalated drinking associated with dependence. As exercise is known to elevate BDNF activity in brain, we conducted pilot studies that have demonstrated exercise attenuates dependence-related escalated ethanol drinking, as well as attenuating the ability of stress to further enhance excessive levels of drinking associated with dependence. With this supportive pilot data, the proposed research plan will build and expand on this work by examining the role of BDNF in the ability of exercise to attenuate stress-enhanced drinking in dependent mice. Specifically, this research project is aimed at utilizing our established stress-ethanol dependence (Stress- CIE) Drinking model to examine whether exercise attenuates stress-enhanced dependence-related excessive drinking via BDNF-TrkB receptor signaling in the dmPFC. Proposed studies will examine whether exercise (wheel-running) blocks stress (FSS)-enhanced escalated drinking in CIE-exposed mice, as well as attenuating reduced BDNF expression in dmPFC that accompanies excessive drinking in the Stress-CIE Drinking model (Aim I). Another set of studies will examine whether direct injection of the TrkB receptor antagonist ANA-12 into the dmPFC blocks the ability of wheel-running to attenuate stress (FSS)-enhanced CIE-induced escalated drinking and whether TrkB receptor knockdown in the dmPFC via siRNA infusion produces a similar effect (Aim II). Finally, studies will examine whether systemic treatment with the flavone derivative and TrkB receptor agonist 7,8-DHF substitutes for exercise in attenuating stress-enhanced CIE-related drinking, and/or whether treatment with 7,8-DHF in the absence of exercise mimics the effects of exercise in the Stress-CIE Drinking model. Additionally, studies will examine whether these exercise-like behavioral effects are due to activation (phosphorylation) of TrkB receptors (pTrkB) and downstream signaling molecules (pERK1/2 and pAKT) in the dmPFC (Aim III). Taken together, this proposal addresses a highly significant and clinically important research topic that is of great relevance to Veteran?s heath care. Results from this research project will generate new findings on mechanisms underlying a potential novel therapeutic approach that addresses a clinically relevant health problem - the exacerbating effects of stress that lead to harmful excessive drinking associated with alcoholism and PTSD-AUD comorbidity - an especially significant problem for our Veterans.